Multiplex diagnostic assays for lyme disease and other tick-borne diseases

ABSTRACT

The present invention provides novel methods of diagnosing and determining treatment strategies for Lyme disease and other tick-borne illnesses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/021,317, filed Mar. 11, 2016, which is the U.S. National Phase ofInternational Application No. PCT/US2014/054972, filed Sep. 10, 2014,which claims priority of U.S. Provisional Application No. 61/877,479filed on Sep. 13, 2013, the contents of which are incorporated herein byreference in their entirety.

GOVERNMENT INTERESTS

This invention was made with government support under grant number R01A1089921 from the National Institutes of Health. The United Statesgovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to the diagnosis of Lyme disease and othertick-borne illnesses.

BACKGROUND OF THE INVENTION

Transmission of pathogens through tick vectors results in differentinfectious diseases in humans with Lyme disease affecting most people inthe United States and Europe. Ticks can infect people withdisease-causing organisms, including three different species of the Lymespirochetes Borrelia burgdorferi, B. afzelii, and B. garinii, theintracellular bacterial pathogen Anaplasma phagocytophilum, and theprotozoan Babesia microti and other Babesia species. Major speciesimplicated in causing Lyme disease are Borrelia burgdorferi sensustricto in the USA and additionally, B. afzelii and B. garinii in theEuropean countries. Co-infection of Borrelia species with two otherpathogens, A. phagocytophilum and Babesia species, has started appearingin both North America and Europe. Babesia species infect red blood cells(erythrocytes), cause babesiosis, and can also be transmitted throughblood transfusion. Recently, several cases of vertical transmission ofB. microti have also been reported. A. phagocytophilum infectspolymorphonuclear leukocytes (PMNs), is an obligate intracellularpathogen and can cause lymphopenia/leukopenia and thrombocytopeniaresulting in human granulocytic anaplasmosis (HGA). Both babesiosis andHGA can be fatal.

Currently, serological tests are used primarily to diagnose all threediseases with culture as the only available confirmatory test. However,assays that detect antibodies do not detect early infections (beforeantibodies are produced), and they cannot distinguish between activeinfections and infections that have been cured by treatment (asantibodies persist long after treatment is completed). Nucleicacid-based assays, on the other hand, are able to specifically detectthe presence of the pathogens that cause Lyme disease and othertick-borne diseases. Yet, current assays that detect specific nucleicacid sequences are insufficiently multiplex to provide an accuratepicture as to whether one or more of the infectious pathogens wereintroduced by the ticks during the bloodmeal. Furthermore, simultaneousinfection with more than one pathogen can affect the sensitivity ofcurrently available tests.

Sensitive diagnostic tests that can accurately diagnose Lyme disease,anaplasmosis and babesiosis are not currently available, thus,emphasizing a need to develop individual test for each pathogen or acombinatorial test for all three tick-borne pathogens to detectco-infection. Thus, there is a desperate need to develop a technicallysimple, rapid and accurate assay to unequivocally diagnose activedisease caused by these three tick-borne infections, individually ortogether.

SUMMARY OF THE INVENTION

The invention addresses the above-mentioned need by providing agents andmethods for diagnosing active disease caused by tick-borne infections.

In one aspect, the invention features multiplex primer-dependent assaymethods, including particularly PCR assay methods, for detectingmultiple genetic targets, including at least two of the following: arecA gene sequence of Borrelia that differs among B. burgdorferi, B.afzelii, and B. garinii; a BmTPK gene sequence of B. microti (conservedin other Babesia species that infect humans); and an APH 1387 genesequence of A. phagocytophilum. The method includes providing a startingamplification reaction mixture that includes, in addition to a human orother mammalian sample suspected to contain at least one target sequenceand amplification reagents (buffer, salts, dNTPs and DNA polymerase),and a primer pair and a molecular beacon probe for each intended target,wherein the primer pair defines an amplification product (“amplicon”)that is 70-300 base pairs in length, and wherein each differentmolecular beacon probe is labeled with a spectrally distinguishablefluorescent or luminescent signaling moiety.

In a second aspect, the invention provides further sensitivity includingamplifying each intended target sequence, if present, by theprimer-dependent (for example, PCR) amplification process and detectingtarget-sequence amplicons with the molecular beacons, in real time, atend point, or by post-amplification thermal analysis of fluorescenceversus temperature, including derivative curves. Assays according tothis invention may be qualitative or quantitative. Preferred embodimentsinclude homogeneous detection.

Methods according to this invention include the foregoing assays whereinthe multiple genetic targets include a human genetic target as control,and wherein the starting amplification reaction mixture includes aprimer pair and a molecular beacon probe for a human DNA gene sequence,and includes detecting amplicon from amplification of said human genesequence. In such methods, a preferred human DNA sequence is a 70-300base-pair region of ACT A1 gene.

Certain preferred embodiments have as targets a BmTPK gene sequence ofB. microti; and an APH 1387 gene sequence of A. phagocytophilum, with orwithout addition of a human gene target sequence.

Certain preferred embodiments have as a target a recA gene sequence ofBorrelia that differs slightly among B. burgdorferi, B. afzelii, and B.garinii, plus at least one target that is a BmTPK gene sequence of B.microti; or an APH 1387 gene sequence of A. phagocytophilum, with orwithout addition of a human gene target sequence. For embodiments havinga recA target sequence, detection may include generatingpost-amplification melting or annealing data to discriminate among B.burgdorferi, B. afzelii, and B. garinii by melting denaturation curve ormelting temperature (Tm).

The present invention includes triplex assays as described above for allthree pathogenic sequences and quadruplex assays that further include ahuman or can include other mammalian genetic target.

In another aspect, the invention features a method, which includesprimer-dependent amplification, preferably PCR amplification, of threetarget sequences of three pathogens: Lyme spirochetes, A.phagocytophilum and Babesia species. Amplification reactions useful inmethods of this invention may be any suitable exponential amplificationmethod, including the polymerase chain reaction (PCR), either symmetricor non-symmetric, digital PCR, the ligase chain reaction (LCR), thenicking enzyme amplification reaction (NEAR), strand-displacementamplification (SDA), nucleic acid sequence-based amplification (NASBA),transcription-mediated amplification (TMA), and rolling circleamplification (RCA). Preferred methods utilize PCR. In nonsymmetric PCRamplification methods, for example asymmetric PCR, one primer, theprimer synthesizing non-target strand, is present in a limiting amountso as to be exhausted prior to completion of amplification, after whichlinear amplification occurs using the remaining primer, the excessprimer that synthesizes the probe binding target strand. A non-symmetricPCR method useful in this invention is LATE-PCR [see, for example,European Patent EP 1,468,114; and Pierce, et al. (2005) Proc Natl AcadSci USA 102:8609-8614]. Preferred methods also include digital PCR [see,for example, Vogelstein and Kinzler (1999) Proc Natl Acad Sci USA98:9236-9241].

The invention also provides PCR methods comprising contacting sample DNAwith a pair of amplification primers for each target sequence, andperforming repeated thermal cycles of primer annealing, primerextension, and strand denaturation (strand melting). Primer annealingmay be performed at a temperature below the primer-extension temperature(for example, three-temperature PCR), or primer annealing and primerextension may be performed at the same temperature (for example,two-temperature PCR). The overall thermal profile of the reaction mayinclude repetitions of a particular cycle, or temperatures/times may bevaried during one or more cycles.

Assay methods according to this invention include detection of amplifiedtarget sequences using fluorescently or luminescently labeledhybridization probes that signal upon hybridization to target sequences,namely the amplification products of the PCR or other amplificationreactions. Suitable probes include TaqMan probes and molecular beaconprobes (“molecular beacons”), which are preferred. Detection may beperformed during the course of amplification (real-time detection) orfollowing amplification (end-point detection) using probes present inthe starting amplification reaction mixture in a single tube, platewell, or other reaction vessel (homogeneous detection). Alternatively,detection may be performed in a melting or annealing step followingamplification. Real-time detection, end-point detection andpost-amplification thermal profiling are preferably performed in ahomogeneous detection assay wherein probes are present in the startingamplification reaction mixture. Then again, using a microfluidic device,amplified products can be moved to a chamber in which they contact oneor more detection probes as well, in some embodiments, or isolatingreagents such as immobilized capture probes.

Primers and probes useful in methods, reaction mixtures and kits of thisinvention are oligonucleotides in the broad sense, by which is meantthat they may be DNA, RNA, mixtures of DNA and RNA, and they may includenon-natural nucleotides (for example, 2′-O-methyl ribonucleotides),non-natural internucleotide linkages (for example, phosphorothioatelinkages), and DNA mimics (for example, PNA or LNA). Both primers andprobes function in part by hybridizing to a sequence of interest in areaction mixture. In the Examples below we utilize primers and probesthat are DNA, which we prefer.

In another embodiment, the present invention further comprises a methodof determining the appropriate treatment regimen by performing the abovemethod and then administering the most effective treatment for thatspecific Borrelia species or other pathogens.

In a further embodiment, the present invention provides an automatedmultiplex diagnostic test for three or more species of Lyme disease.

It further provides a method of for accurate diagnosis of the disease(s)and then determining the appropriate treatment regimen for the specificcausative pathogen(s), bacteria or eukaryotic parasite.

Another embodiment of the present invention provides an automatedmultiplex diagnostic test for simultaneous detection of all threeprevalent species of Lyme disease spirochetes.

This invention includes reagent mixtures for performing methodsaccording to this invention, as well as kits of reagents for preparingsuch reaction mixtures and for performing such methods. In one example,a kit for diagnosing a tick-borne disease comprises (i) a first pair ofprimers and a first molecular beacon probe for a first target selectedfrom a group consisting of a recA gene sequence of Borrelia that differsamong B. burgdorferi, B. afzelii, and B. garinii; a BmTPK gene sequenceof B. microti; and an APH 1387 gene sequence of A. phagocytophilum, and(ii) a second pair of primers and a second molecular beacon probe for asecond target selected from said group. The primer pairs defineamplicons that are 70-300 base pairs in length, and each molecularbeacon probe is labeled with a spectrally distinguishable fluorescent orluminescent signaling moiety, The kit can include a third pair ofprimers and a third molecular beacon probe for a human DNA genesequence. In some embodiments, the first or second pair of primers canbe selected from the group consisting of SEQ ID Nos. 10 and 11, SEQ IDNos. 12 and 13, SEQ ID Nos. 14 and 15, SEQ ID Nos. 18 and 19, SEQ IDNos. 24 and 25, and SEQ ID Nos. 26 and 27. The first or second molecularbeacon probe can be selected from the group consisting of SEQ ID Nos. 5,16, and 20. The human DNA gene can be the ACT A1 gene and in that case,the third pair of primers can have the sequences of SEQ ID Nos. 21 and22, respectively, and the third molecular beacon probe can have thesequence of SEQ ID No. 23. The invention can also include slightmodification of the normal molecular probes within the said ampliconsspecific for each Borrelia species tagged with different fluorophore todistinguish different species of these pathogens or can include thesloppy molecular beacons within the region for detection [see, forexample, European Patent EP 1 921 169], such as the RecA3 probedescribed below.

Preferred reagent mixtures and kits according to this invention includebuffers, salts, dNTPs, DNA polymerase, primers for the intended targets,and molecular beacon probes for the intended target sequences.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects and advantages of theinvention will be apparent from the description and the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C are a set of graphs showing (A) real-timefluorescence (fluorescence intensity versus cycle number) for the RecA3probe for different starting concentrations (10⁶ to 10⁰) of B.burgdorferi target in the assay of Example 1; (B) PCR threshold cycleversus starting concentration of B. burgdorferi from FIG. 1A; and (C)real-time fluorescence for the ACTA1 probe in the assay of Example 1.

FIGS. 2A and 2B are a set of graphs showing (A) hybridization melting(fluorescence intensity versus temperature) of the RecA3 probe when theassay of Example 2 was performed with conserved oligonucleotides in allthree Borrelia species: B. burgdorferi, B. afzelii, and B. garinii. and(B) melting curves (−dF/dT) of products of an asymmetric PCR using RecF3and RecR3 primers and RecA3 molecular beacon with three Borreliaspecies, B. burgdorferi, B. afzelii and B. garinii genomic DNA astemplate.

FIGS. 3A, 3B and 3C are a set of graphs showing (A) real-timefluorescence (fluorescence intensity versus PCR cycle number) for theBmTPK probe for different starting concentrations (10⁶ to 10⁰) of B.microti target in the assay of Example 3; (B) PCR threshold cycle versusstarting concentration of B. microti from FIG. 3A; and (C) real-timefluorescence for the ACTA1 probe in the assay of Example 3.

FIGS. 4A, 4B and 4C are a set of graphs showing (A) real-timefluorescence (fluorescence intensity versus PCR cycle number) for theAph1387 probe for different starting concentrations (10⁶ to 10°) of A.phagocytophilum target in the assay of Example 4; (B) PCR thresholdcycle versus starting concentration of A. phagocytophilum from FIG. 4A;and (C) real-time fluorescence for the ACTA1 probe in the assay ofExample 4.

FIGS. 5A, 5B, 5C and 5D are a set of graphs showing (A) of real-timefluorescence (fluorescence intensity versus PCR cycle number) for theRecA3 probe for different starting concentrations (10⁶ to 10°) of B.burgdorferi target in the assay of Example 5; (B) real-time fluorescence(fluorescence intensity versus PCR cycle number) for the BmTPK probe fordifferent starting concentrations (10⁶ to 10°) of B. microti target inthe assay of Example 5; (C) real-time fluorescence (fluorescenceintensity versus PCR cycle number) for the Aph1387 probe for differentstarting concentrations (10⁶ to 10°) of A. phagocytophilum target in theassay of Example 5; and (D) real-time fluorescence for the ACTA1 probein the assay of Example 5.

FIG. 6 is a graph of fluorescence intensity versus PCR cycle number forthree probes in a triplex amplification starting with 10⁶ copies of B.burgdorferi genomic DNA (including 10⁶ copies of the recA gene) mixedwith genomic copies of each of A. phagocytophilum and B. microti genomicDNA reflecting 10³ copies of the BmTPK and APH1387 genes in the assaysof Example 6.

FIG. 7 is a graph of fluorescence intensity versus PCR cycle number forthree probes in a triplex amplification starting with 10 copies of B.burgdorferi genomic DNA (including 10 copies of the recA gene) mixedwith copies of each of A. phagocytophilum and B. microti genomic DNAreflecting 10³ copies of the BmTPK and APH1387 genes in the assays ofExample 7.

DETAILED DESCRIPTION OF INVENTION AND EMBODIMENTS

The invention is based, at least in part, on unexpected discoveries thatmolecular beacon probes-based real-time polymerase chain reaction (PCR)can be used to diagnose Lyme disease, anaplasmosis and babesiosis in asensitive and specific manner. Since microbial nucleic acids do notpersist much longer after cure of a disease, PCR-based assays are idealfor detection of these three pathogens. As disclosed herein, theeffective combinations of primers enable the amplification of targetsequences specific to each infectious agent. In addition, the design oftarget-specific probes that are labeled with, e.g., differently coloredfluorophores, in combination with the primers for each infectious agentallows one to unequivocally diagnose active disease caused by differenttick-borne infections, individually or together. Before this invention,no one has yet been able to design and demonstrate the function of thedesired multi-species combinations for testing blood samples frompatients and for testing donated blood.

Due to the presence of nucleases in the serum, nucleic acids of thepathogens do not persist in the host much longer after the disease iscured [see Kurreck (2003), Eur J Biochem./FEBS 270:1628-1644; Meng etal. (2011) BMC Biotechnol 11:88; and Mutwiri et al. (2004) J. ControlRelease 97:1-17]. Therefore, PCR assays can be used as test of cure forvarious diseases. Selection of proper PCR target and conditions alongwith the use of efficient detection probe are critical for developmentof sensitive and specific diagnostic assays.

Molecular beacons are hairpin-shaped oligonucleotide probes that can bedesigned to be highly specific for their target sequences and can belabeled with distinguishably colored fluorophores [Marras et al. (2002)Nucleic Acids Res 30:e122]. The single-stranded loop of highly specificmolecular beacons is designed to be complementary to a unique genesequence that identifies the infectious agent. Drs. Marras, Tyagi, andKramer used these probes to distinguish alleles that differ by as littleas a single nucleotide polymorphism [Marras et al. (1999) Genet Anal14:151-156; Piatek et al. (1998) Nat Biotechnol 16:359-63; Tyagi et al.(1998) Nat Biotechnol 16:49-53; and Tyagi et al. (2000) Nat Biotechnol18:1191-1196]. The basis of this extraordinary specificity is thathairpin-shaped probes can assume two different stable states, by: (i)forming double-stranded hybrids with their target sequence, or (ii)retaining their partially double-stranded structure when not bound to atarget. Any mismatch between the probe sequence of the molecular beaconand the target sequence destabilizes the probe-target hybrid, leading toreturn of the molecular beacon in its stable hairpin structure [Bonnetet al. (1999) Proc Natl Acad Sci USA 96:6171-6176; and Mhlanga andMalmberg (2001) Methods 25:463-471]. Thus, molecular beacon probes areinherently more specific than linear TaqMan probes, which are morestable when bound to their target, whether or not they are fullycomplementary to the target [Bonnet et al. (1999) Proc Natl Acad Sci USA96:6171-6176; Petersen et al. (2004) Mol Cell Probes 18:117-122; andTapp et al. (2000) Biotechniques 28:732-738]. The specificity ofmolecular beacon probes to detect the recA gene of B. burgdorferi, andto quantitate the number of spirochetes present in infected mouse tissuewas previously reported [Saidac et al. (2009) BMC Microbiol 9:43-52].

The present invention involves assays that work in the presence of humanDNA, such that they are useful as a diagnostic test for human Lymedisease. The methods for diagnosing active disease of the presentinvention detect three major Lyme spirochete species, B. burgdorferisensu stricto, B. afzelii, and B. garinii in the same real time-PCRassay. An alternative aspect of the invention separate PCR assays areutilized by selecting the sequence-specific molecular beacon probes foreach species. This present invention includes highly sensitive andmultiplex real-time-PCR (rt-PCR) assay methods using target-specificmolecular beacons that can distinguish B. burgdorferi, A.phagocytophilum and B. microti simultaneously in the same assay.

Certain embodiments of assays of this invention employ real-time PCRamplification with homogeneous detection of target DNA sequence of eachof these three infectious-organism targets. The assays can be extendedto include reverse transcription for detection of RNA, if needed.Previously, TaqMan probes developed by Applied Biosystems, which aresingle stranded oligonucleotides labeled with a fluorophore-quencherthat hybridize with the sequence present in the internal region of anamplified PCR product have been used for detection of Lyme spirochetesin ticks and mammals. When free in solution, TaqMan probes form randomcoils in which fluorophore reporter and quencher come in closeproximity, enabling Fluorescence Resonance Energy Transfer (FRET) fromthe fluorophore to the quencher. This mechanism alleviates thefluorescence signal of the reporter. In the presence of the target,TaqMan probe-target hybrid comes in contact with the Taq Polymeraseduring the extension phase of PCR cycles. Inherent 5′exonucleaseactivity of the enzyme then cleaves the probe, releasing the fluorescentreporter from the portion of the probe that includes the quencher. Thisleads to increase in the fluorescence intensity at each PCR cycle sinceFRET cannot occur anymore. Random coil formation of the free TaqManprobes sometimes results in only partial quenching of fluorescence inthe absence of the specific target. Thus, TaqMan probes have not proveneffective in diagnosing active disease caused by tick-borne pathogens.

This invention employs molecular beacons, which are dual fluorescentlylabeled single-stranded oligonucleotide probes that form stem-and-loopstructures, such that the target-recognition sequence is located,entirely or predominantly, in the loop region and complementary terminalsequences (arms) form a stem bringing the fluorophore and quencher intoclose proximity [Marras et al. (1999) Genet Anal 14:151-156; Mazepa etal. (2010) J Am Anim Hosp Assoc 46:405-412; Tyagi et al. (2000) NatBiotechnol 18:1191-1196; and Vannier et al. (2008) Infectious DiseaseClinics of North America 22:469-488, viii-ix]. The quenching offluorescence by contact is highly efficient and exhibits minimalbackground fluorescence in the absence of target sequences. Thetechnology previously described for the use of molecular beacons asprobes for PCR amplification products (amplicons) is significantlyimproved upon for this invention. Multiple molecular beacons can belabeled with different fluorophores, and several different probes can beused simultaneously in multiplex assays. Quenching of signal in theabsence of the target is much more pronounced when molecular beacons areused as probes resulting in minimal background fluorescence when theprobes are present in solution. In addition, molecular beacons can bedesigned to successfully discriminate single nucleotide polymorphisms(SNPs). Preferred assays according to this invention distinguish variousLyme spirochete species that show SNPs in the PCR-amplified region ofthe recA gene.

Previously, a 222-bp amplicon from recA gene of B. burgdorferi usingRecF (forward) and RecR (reverse) primers was amplified in a real-timePCR assay using SYBR Green DNA dye for spirochete quantitation. In thepresent invention the same primers were used with a Borrelia-specificmolecular beacon probe designated “RecA3”, whose sequences are all givenin Table 1 below. The recA sequence defined by the RecF and RecR primersis:

(SEQ ID No. 1) 5′GTGGATCTATTGTATTAGATGAGGCTCTCGGCATTGGCGGATATCCTAGGGGGCGCATAATAGAAATTTTTGGCCCCGAGTCGTCTGGCAAGACTACTTTAACTCTTCAAGCGATTGCTGAGGTGCAAAAAGAAGGTGGGATAGCTGCTTTTATTGATGCTGAGCATGCTCTTGATCCTGTTTATGCAAAAGCTTTAGGTGTTAATGTTGCAGAACTTTGGC3′

Three sequences are underlined: the sequence of the RecF primer, thesequence complementary to the RecR primer (SEQ ID No. 17), and thesequence of the molecular beacon RecA3 probe's target-recognitionsequence (which in this case is the loop sequence plus multiplenucleotides of each arm) (bold-underlined). Presented below is thesequence of the complementary strand of B. burgdorferi in the region towhich the RecA3 probe binds, plus the corresponding sequences of B.afzelii and B. garinii that are complementary to the loop plusadditional stem-nucleotides of the RecA3 probe, which is shown in 3′5′direction for visualizing hybridization and mismatch.

B. burgdorferi (SEQ ID No. 2) 5′ TTAT GCGCCCCCTAGGATATCCGCCA ATGC 3′B. afzelii (SEQ ID No. 3) 5′ TTAT GCGCCCCCTAGGATATCCACCA ATGC 3′B. garinii (SEQ ID No. 4) 5″ TTAT TCGCCCCCTAGGATATCCACCA ATGC 3′RecA3 probe (SEQ ID No. 5)   3′ GAC CGCGGGGGATCCTATAGGCG GTC 5′

In the above sequences, spaces have been left before and after theregions of complementarity for illustrative purposes. As indicated, themolecular beacon probe (SEQ ID No. 5) includes a probe sequenceconsisting of the loop (SEQ ID No. 30) and three nucleotides of each arm(bold) that is perfectly complementary to the B. burgdorferi sequence(SEQ ID No. 2). The probe is complementary to, but not perfectlycomplementary to, the other species, particularly the target sequencesof B. afzelii (SEQ ID No. 3) and B. garinii (SEQ ID No. 4), whichpossess one (B. afzelii) or two (B. garinii) single nucleotidepolymorphisms (SNPs) facilitating differentiation of species by post-PCTTm determination. Target nucleotides that are mismatched from the probesequence are bolded and underlined. Probe nucleotides forming the stemare underlined.

As indicated above, the RecF and RecR primers define a 222 base-pair(bp) amplicon that includes the sequence probed by the RecA3 probe. Aswill be appreciated, other primers could be chosen to produce anamplicon including that sequence, as by sliding the current primersalong the sequence of the recA gene. Primer design is well known andtakes into account the amplification mixture and protocol intended to beused. Similarly, the loop sequence of a probe intended to be perfectlycomplementary to the B. burgdorferi species in the region of interestcould be varied by sliding the loop along the sequence of the genesequence or by changing its length. Once again the amplificationparameters intended to be used are taken into account. The “BeaconDesigner™” computer program often suggests multiple loop sequences tochoose from.

The RecA3 molecular beacons probe is labeled with a fluorescent moietyon one end (in the embodiment used in the Examples a Fluorescein (FAM)reporter molecule at the 5′ terminus) and a non-fluorescent quenchermoiety on the other end (in the embodiment used in the Examples a BlackHole Quencher 1 (BHQ-1) at the 3′ terminus). As will be appreciated,other fluorescent or luminescent moieties could be used as labels, ascould different non-fluorescent quencher moieties. Molecular beaconRecA3 has been shown to be highly efficient and sensitive for thedetection and quantification of B. burgdorferi in the infected mammalian(mouse) tissues by real-time PCR. Preferred assays according to thisinvention include also amplifying a human target DNA sequence as apositive control to employ with the human samples. In the Examples, weutilize for this purpose primers and a probe for a target sequence inthe single-copy Act A1 gene. In the embodiment utilized in the Examplesthe molecular beacon probe, designated ACTA1, is labeled on one end witha fluorescent moiety and on the other end with a non-fluorescentquencher moiety (in the Examples a Quasar 670 fluorophore and Black HoleQuencher 2 (BHQ-2) quencher. This human PCR target and probe provide apositive control to determine the quality of DNA isolated from humanpatient samples.

The 325 bp ACT A1 amplicon is derived from exon 3 (Accession No.NG006672) and the 104 nucleotide sequence obtained by using 5ACTA1 and3ACTA1 primers is:

(SEQ ID No. 6) 5′AGAGCAAGAGAGGTATCCTGACCCTGAAGTACCCTATCGAGCACGGCATCATCACCAACTGGGATGACATGGAGAAGATCTGGCACCACACCTT CTACAACGAG3′

Three sequences are underlined: the sequence the 5ACTA1 primer, thesequence complementary to the 3ACTA1 primer, and the sequence of theACTA1 molecular beacon's (SEQ ID No. 23) probe sequence, which in thiscase is the loop sequence (bold-underlined, SEQ ID No. 28).

The Act A1 gene-segment target can be used in multiplex assays to detectB. burgdorferi DNA in the presence of human DNA. Indeed, sensitivity ofdetection of B. burgdorferi remained unaffected in the multiplex assaysrelative to that in the monoplex assay when B. burgdorferi DNA alone ispresent.

The primers for the B. microti TPK gene, the A. phagocytophilum APH 1387gene, and the human ACT A1 gene were chosen to target regions thatdistinguish the respective organisms but are believed to be quiteconserved regions among species and strains of the respective targets.Amplification conditions were taken into account, as all primer pairswere intended to amplify efficiently in a single multiplex assay.Molecular beacon probes for these targets can be designed using theBeacon Designer™ computer program, so that they too would work in asingle multiplex assay.

Certain assays according to this invention amplify and detect a Borreliatarget sequence that differs among B. burgdorferi, B. afzelii, and B.garinii. Embodiments of such assays can utilize three differentlycolored Borrelia molecular beacon probes: one allele-discriminatingprobe that is perfectly complementary to each species [see, Tyagi et al.(1998) Multicolor molecular beacons for allele discrimination. NatBiotechnol 16:49 53]. Preferred embodiments utilize a singlemismatch-tolerant probe that hybridizes to all three species with adetectably distinct melting temperature (Tm) for each species. In onepreferred embodiment the probe's target-complementary sequence isperfectly complementary to one species (particularly for assays intendedfor use in the USA., that species is B. burgdorferi sensu stricto,whereas for assays intended for use in Europe, those species are B.burgdorferi, B. afzelii, and B. garinii). Assays utilizing a singleBorrelia probe are simpler to manufacture, and they utilize less colorspace of an instrument. As shown in Example 2 below, using the RecA3probe, melting curves were able to distinguish among three species. Apost-amplification melting or annealing curve can potentially be used toidentify which species is present in a sample, if the Tm's are at least3° C., preferably at least 5° C., apart. In one embodiment a differentset of primers that are perfectly conserved in all three Borreliaspecies was used to obtain a slightly longer, 287 bp size amplicon andthe same, RecA3 molecular beacon in the assay. The target sequence forthe B. burgdorferi is given below:

(SEQ ID No. 7) 5′GCAAGAGTTCAAATAGAAAAAGCTTTTGGAAAGGGAAGTCTTATTAAGATGGGGGAATCTCCTGTTGGACAAGGTATAAAAAGTATGTCAAGTGGATCTATTGTATTAGATGAGGCTCTCGGCATTGGCGGATATCCTAGGGGGCGCATAATAGAAATTTTTGGCCCCGAGTCGTCTGGCAAGACTACTTTAACTCTTCAAGCGATTGCTGAGGTGCAAAAAGAAGGTGGGATAGCTGCTTTTATTGATGCTGAGCATGCTCTTGATCCTGTTTATGCAAAAGCTTT3′

Two sequences are underlined: the RecF3 primer and the sequencecomplementary to RecR3 primer. Nucleotides depicting the RecA3 probe aremarked by bold letters.

Embodiments of assays according to this invention are multiplex assaysthat include detection of B. microti and A. phagocytophilum in additionto Borrelia. For the parasite B. microti (and other Babesia species) wedisclose a preferred embodiment of PCR primers 5BmTPK and 3BmTPK foramplifying a 141-base-pair (bp) sequence of the BmTPK gene (AccessionNo. F0082871), using the primers and a molecular beacon probe specificfor that sequence, which is as follows:

(SEQ ID No. 8) 5′TGAGAGGAACGACCATAGCCTTTTACATATGACACAAGCTATAACTATAGCAGAAAATGGAATTTCGGTGTTGTTGACCAGCGGCCGCGAAGAAGGATGGCCAATTTTTCCAAGACATTTTTCGTGTGATTTACCTGATGG3′

Three sequences are underlined: the 5BmTPK primer, the sequencecomplementary to the 3BmTPK1 primer, and the BmTPK probe's loop sequence(bold-underlined). Note that two additional probe nucleotides, TClocated 5′ to the loop sequence, are complementary to the targetsequence.

For A. phagocytophilum we disclose a preferred embodiment of PCR primersfor amplifying a 152 bp sequence of the APH1387 gene (Accession No.CP000235), obtained by using 5Aphagocyt and 3Aphagocyt primers andAPH1387 molecular beacon probe specific for that sequence. The sequenceof the amplicon is:

(SEQ ID No. 9) 5′ATGGCTACTACGAAGGATGTGCTTGTGACAAAGATGCCAGCACTAATGCGTACTCGTATGACAAGTGTAGGGTAGTACGGGGAACGTGGAGACCGAGCGAACTGGTTTTATATGTTGGTGATGAGCATGTGGCATGTAGAGA TGTTGCTTCG3′

Three sequences are underlined: the 5Aphagocyt primer, the sequencecomplementary to the 3Aphagocyt primer, and the APH1387 probe's loopsequence (bold-underlined, SEQ ID No. 29).

Both of the foregoing primer pairs and the probe are adapted to workwith the Borrelia primers and probe in a real-time or end-pointmultiplex PCR assay. The present invention demonstrates that a multiplexPCR assay for all three targets can also include primers and a probe forhuman DNA. Furthermore, our single quadruplex assay is able todistinguish all three major Borrelia species implicated in Lyme diseasein humans. Additionally, by including primers and a probe for human DNA,assays according to this invention are quantitative for the startinglevels of the pathogenic targets.

The present invention provides a novel multiplex assay method todiagnose three tick-borne illnesses. It comprises a sensitive, specificand user-friendly method of diagnosing Lyme disease, as well astick-borne pathogens comprising A. phagocytophilum and Babesia speciesby obtaining a biological sample from a human or other mammal anddetecting the DNA of one or more of the three major Lyme spirochetes andat least one of A. phagocytophilum and Babesia species, preferably both.Preferred embodiments also include detecting human DNA as a positivecontrol that determines quality of DNA and permits quantitation ofresults. Our most preferred assay is a quadruplex PCR assay thatutilizes a single Borrelia probe having distinguishable Tm's against thethree Borrelia species. Alternatively, once a determination is maderegarding presence of Borrelia in the sample in the amplification partof the multiplex assay, a follow up assay can be performed to identifythe specific Borrelia species using a complement of primers, forexample, species-specific primers, and molecular beacon probes, forexample, species-specific probes, whose colors or color identify whichspecies is present. A further alternative, utilizing species-specificBorrelia probes as stated above, would include five molecular beaconprobes, or six, if a human DNA target such as the Act A1 gene isincluded. The above method further provides a basis of determining theappropriate treatment and then employing the most effective treatmentregimen for that specific pathogen, especially during the latepersistent disease.

The present invention resolves a serious unmet need in the diagnosis andprognosis of Lyme disease, anaplasmosis and babesiosis. Current serologybased tests for these diseases cannot distinguish whether the patientformerly had the disease or is still infected. Furthermore, reinfectioncannot be detected using the serological tests, which is of particularimportance in the endemic regions. The present invention will be able todiagnose the active disease phase and will be able to classify theinfection based upon the particular tick-borne pathogen.

Nucleic acid-based diagnostic tests for infectious diseases are becomingincreasingly useful. Applicants previously developed and assessed areal-time PCR based test that incorporated specific molecular beaconprobes against the recA gene of Lyme disease-causing Borreliaburgdorferi to detect and quantify this spirochete in infected mousetissues [Saidac et al. (2009) BMC Microbiol 9:43-52]. The presentinvention converts the test for patients and provides a sensitive,specific and accurate test for diagnosis of Lyme disease in the endemicregions of both the United States and Europe and certain regions of Asiawhere the disease is prevalent. This assay can detect two additionaltick-borne emerging pathogenic agents responsible for devastating andoften fatal diseases, anaplasmosis and babesiosis, along with Lymedisease-causing Borrelia species simultaneously.

The present invention represents several advantages over the currentscience, specifically with the use of real time-PCR (rt-PCR) andspecific probes. This method offers a decrease in turn-around time,increased reliability and efficiency, and greater accuracy. In addition,automation of nucleic acid extraction coupled with rt-PCR results in afast and accurate platform for diagnosis and a closed system preventscross-contamination. Finally, internal positive and negative controls(sample without template) can ensure good quality of the preparedsamples, confirm the sensitivity and accuracy of the test, and ensurelack of contamination. Importantly, this aspect of the present inventionprovides a method of selecting an appropriate treatment and determiningthe efficacy of the administered therapy in a timely manner.

The assay of the present invention can be optimized to detect thepresence of the DNA for a sensitive and specific diagnosis of the activeLyme disease. Therefore, it is expected that the subjects, especiallythose exhibiting persistent manifestations will directly benefit fromthe study. Furthermore, use of the sequence specific molecular beaconprobes designed for the selected specific real-time polymerase chainreaction amplicon for each tick-borne pathogen and labeled withdifferent fluorophores will lead to the development of a very sensitive,specific and confirmatory diagnostic assay for single or multipletick-borne co-infecting pathogens. Already standardized recA ampliconsand specific molecular beacons can potentially be used to distinguishthree species of Lyme spirochetes in this assay. In addition, a PCRamplicon of APH1387 gene, which encodes a unique A. phagocytophilumprotein essential for its pathogenesis and unique region of theTPK-encoding gene of Babesia species and respective sequence specificmolecular beacons designed and optimized for these amplicons can beincluded in the multiplex assay. Hence, a single test will be able toidentify the presence of one or more tick-borne pathogens in thepatients. Although molecular beacon probes have been used for diagnosisof some diseases, such as tuberculosis, such a test for Lyme disease andother tick-borne emerging pathogens does not exist. This test will notonly be more sensitive and specific for the active disease, it willultimately diagnose multiple tick-borne diseases simultaneously.

In one embodiment, the present invention comprises a sensitive, specificand user-friendly method of diagnosing Lyme disease, even detecting theDNA of one or more of the three major Lyme spirochetes as well astick-borne pathogens comprising A. phagocytophilum and Babesia from thesame biological sample from a mammal. It provides the distinct advantageover the current methods, as it comprises a single test and can diagnosemultiple tick-borne diseases simultaneously within a few hours. Inaddition, the assay will detect both bacterial and parasitic pathogenscommonly present (B. burgdorferi that causes Lyme disease) or emergingA. phagocytophilum that causes Human Granulocytic Anaplasmosis, i.e.,HGA, and Babesia species that causes babesiosis, respectively.

In another embodiment, the present invention further comprises a methodof determining the appropriate treatment regimen by performing the abovemethod and then administering the most effective treatment for thatspecific spirochete. Since treatment strategies are different forbacterial and parasitic pathogens, a simultaneous and accurate detectionof the pathogen will help design better treatment regimes for theco-infections with the emerging tick-borne pathogens, especially for thepatients in the endemic regions of the United States and Europe.

In a further embodiment, the present invention provides an automatedmultiplex diagnostic test for three or more species of Lyme disease.

It further provides a method of for accurate diagnosis of the disease(s)and then determining the appropriate treatment regimen for the specificcausative pathogen(s), bacteria or eukaryotic parasite.

Another embodiment of the present invention provides an automatedmultiplex diagnostic test for simultaneous detection of all threeprevalent species of Lyme disease spirochetes and their differentiation.

Another embodiment of the present invention is selection of the specificsegments of genes of A. phagocytophilum and Babesia microti, twoemerging tick-borne pathogens, Aph1387 and thiamine pyrophosphokinase(TPK) respectively and design of the primers and molecular beacon probesfor their amplification and detection at the conditions that are usedfor amplification of recA and ACT A1 amplicons of B. burgdorferi andhumans, respectively. Molecular beacon probes for Aph1387 amplificationproduct are labeled with fluorophore CAL Fluor Red 610 with BHQ-2 asquencher and for TPK is labeled with CAL Fluor Orange 560 with BHQ-1quencher. Both of these gene segments and fluorophores in molecularbeacons are compatible for each other and a single multiplex PCR candetect the presence of DNA of each pathogen.

Another aspect of this invention is that an accurate and sensitive testfor Babesia species will allow testing of blood in the blood banks toavoid transmission of this fatal disease through blood transfusion. Asdocumentation of the cases of blood transfusion-associated babesiosisand resulting deaths have started appearing in the last few years, thereis urgency to detect the parasite in the donated blood to prevent suchtransmission. Our assay conducted with the original blood samples orfollowing blood culture in vitro provides an easy and cost-effectivemechanism to achieve this objective.

One of the most important features of this invention is that design ofthe primers and molecular beacon probes and selection of thefluorophores for molecular beacons that are compatible with each otherand do not exhibit noticeable interference. Furthermore, the optimizedPCR conditions are such that amplification of gene segments, probehybridization and detection of all four amplicons is possible in thesame reaction. There is almost no competition in the PCR and probehybridization as demonstrated by the sensitivity and specificity ofdetection of each target DNA according to their quantity present in bothmonoplex and multiplex assays. Thus, we have discovered that a singlemultiplex assay can both detect the presence of each target DNA and canalso accurately quantify it. Although infections with bacterialpathogens, B. burgdorferi and A. phagocytophilum, can be treated withthe same antibiotics, treatment of parasitic disease babesiosis requiresdifferent drug regime. Since the subjective symptoms for thesetick-borne illnesses may overlap, a single assay that can discriminatethe presence of one or more of these pathogens from this invention willallow appropriate treatment of the patients in a timely manner.

A “nucleic acid” refers to a DNA molecule (e.g., a cDNA or genomic DNA),an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNAanalog can be synthesized from nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

As used herein, the term “target nucleic acid” or “target sequence”refers to a nucleic acid containing a target nucleic acid sequence. Atarget nucleic acid may be single-stranded or double-stranded, and oftenis DNA, RNA, a derivative of DNA or RNA, or a combination thereof. A“target nucleic acid sequence,” “target sequence” or “target region”means a specific sequence comprising all or part of the sequence of asingle-stranded nucleic acid. A target sequence may be within a nucleicacid template, which may be any form of single-stranded ordouble-stranded nucleic acid.

As used herein the term “amplification” and its variants includes anyprocess for producing multiple copies or complements of at least someportion of a polynucleotide, said polynucleotide typically beingreferred to as a “template.” The template polynucleotide can be singlestranded or double stranded. A template may be a purified or isolatednucleic acid, or may be non-purified or non-isolated. Amplification of agiven template can result in the generation of a population ofpolynucleotide amplification products, collectively referred to as an“amplicon.” The polynucleotides of the amplicon can be single strandedor double stranded, or a mixture of both. Typically, the template willinclude a target sequence, and the resulting amplicon will includepolynucleotides having a sequence that is either substantially identicalor substantially complementary to the target sequence. In someembodiments, the polynucleotides of a particular amplicon aresubstantially identical, or substantially complementary, to each other;alternatively, in some embodiments the polynucleotides within a givenamplicon can have nucleotide sequences that vary from each other.Amplification can proceed in linear or exponential fashion, and caninvolve repeated and consecutive replications of a given template toform two or more amplification products. Some typical amplificationreactions involve successive and repeated cycles of template-basednucleic acid synthesis, resulting in the formation of a plurality ofdaughter polynucleotides containing at least some portion of thenucleotide sequence of the template and sharing at least some degree ofnucleotide sequence identity (or complementarity) with the template. Insome embodiments, each instance of nucleic acid synthesis, which can bereferred to as a “cycle” of amplification, includes creating free 3′ end(e.g., by nicking one strand of a dsDNA) thereby generating a primer andprimer extension steps; optionally, an additional denaturation step canalso be included wherein the template is partially or completelydenatured. In some embodiments, one round of amplification includes agiven number of repetitions of a single cycle of amplification. Forexample, a round of amplification can include 5, 10, 15, 20, 25, 30, 35,40, 50, or more repetitions of a particular cycle. In one exemplaryembodiment, amplification includes any reaction wherein a particularpolynucleotide template is subjected to two consecutive cycles ofnucleic acid synthesis. The synthesis can include template-dependentnucleic acid synthesis.

The term “primer” or “primer oligonucleotide” refers to a strand ofnucleic acid or an oligonucleotide capable of hybridizing to a templatenucleic acid and acting as the initiation point for incorporatingextension nucleotides according to the composition of the templatenucleic acid for nucleic acid synthesis. “Extension nucleotides” referto any nucleotides (e.g., dNTP) capable of being incorporated into anextension product during amplification, i.e., DNA, RNA, or a derivativeif DNA or RNA, which may include a label.

The term “probe” as used herein refers to an oligonucleotide capable ofbinding to a target nucleic acid of complementary sequence through oneor more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. Probes may bind targetsequences lacking complete complementarity with the probe sequencedepending upon the stringency of the hybridization conditions. There maybe any number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids described herein. However, if the number of mutations isso great that no hybridization can occur under even the least stringentof hybridization conditions, the sequence is not a complementary targetsequence. A probe may be single stranded or partially single andpartially double stranded. The strandedness of the probe is dictated bythe structure, composition, and properties of the target sequence.Probes may be directly labeled or indirectly labeled with a label suchas fluorophore or biotin to which a streptavidin complex may later bind.

“Complement” or “complementary” as used herein to refer to a nucleicacid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen basepairing between nucleotides or nucleotide analogs of nucleic acidmolecules. A full complement or fully complementary may mean 100%complementary base pairing between nucleotides or nucleotide analogs ofnucleic acid molecules.

“Hybridization” or “hybridize” or “anneal” refers to the ability ofcompletely or partially complementary nucleic acid strands to cometogether under specified hybridization conditions (e.g., stringenthybridization conditions) in a parallel or preferably antiparallelorientation to form a stable double-stranded structure or region(sometimes called a “hybrid”) in which the two constituent strands arejoined by hydrogen bonds. Although hydrogen bonds typically form betweenadenine and thymine or uracil (A and T or U) or cytosine and guanine (Cand G), other base pairs may form [e.g., Adams et al. (1992) TheBiochemistry of the Nucleic Acids, 11th ed.].

As used herein, the term “contacting” and its variants, when used inreference to any set of components, includes any process whereby thecomponents to be contacted are mixed into same mixture (for example, areadded into the same compartment or solution), and does not necessarilyrequire actual physical contact between the recited components. Therecited components can be contacted in any order or any combination (orsubcombination), and can include situations where one or some of therecited components are subsequently removed from the mixture, optionallyprior to addition of other recited components. For example, “contactingA with B and C” includes any and all of the following situations: (i) Ais mixed with C, then B is added to the mixture; (ii) A and B are mixedinto a mixture; B is removed from the mixture, and then C is added tothe mixture; and (iii) A is added to a mixture of B and C. For example,“contacting a template with a reaction mixture” includes any or all ofthe following situations: (i) the template is contacted with a firstcomponent of the reaction mixture to create a mixture; then othercomponents of the reaction mixture are added in any order or combinationto the mixture; and (ii) the reaction mixture is fully formed prior tomixture with the template.

Examples

The present invention is described more fully by way of the followingnon-limiting experimental examples. Modifications of these examples willbe apparent to those skilled in the art and are intended to be withinthe scope of the invention, as described.

Materials and Methods

Microbial strains and human cell line. For standardization of conditionsfor the diagnostic assay for Lyme disease, N40 strain clone D10/E9, ofB. burgdorferi (sensu stricto), VS461 strain of B. afzelii and PBistrain of B. garinii were grown in BSKII medium supplemented with 6%rabbit serum at 33° C. (E. Vannier of Tufts Medical Center at Boston,and E. Fikrig of Yale University School of Medicine provided the genomicDNA from B. microti strain RM/NS and A. phagocytophilum strain HZ,respectively.) Human embryonic kidney 293 cells were cultured in a 1:1mix of DMEM (low glucose) and Ham's F12 medium (Invitrogen, CA)supplemented with 10% FBS to isolate DNA for the assays.

Isolation of B. burgdorferi and human genomic DNA. Total genomic DNA wasisolated from the Lyme spirochetes grown to a density of ˜10⁸spirochetes/ml using the protocol we described previously [Parveen andLeong (2000) Mol Microbiol 35:1220-1234]. DNA from 293 cells wasisolated using the previously described protocol [Morrison et al. (1999)J Clin Microbiol 37:987-992) with two modifications. First,PLG-containing tubes (Qiagen Sciences, MD) were used for phenol andchloroform extraction, since they allow clean separation of the topaqueous layer by decantation after centrifugation. Second, a final stepof passing the DNA through DNeasy kit columns (Qiagen Sciences) wasincluded to obtain good quality DNA for rt-PCR.

Molecular beacons design. Design of molecular beacon probe to hybridizeto the recA gene of Lyme spirochetes and tagged with FAM fluorophore andBHQ-1 quencher were described previously [Saidac et al. (2009) BMCMicrobiol 9:43-52]. Other molecular beacon probes were designed usingthe previously described strategies such that fluorophore emissionprofiles are non-overlapping [Vet and Marras (2005) Methods Mol Biol288:273-290]. Briefly, molecular beacon probes for; ACTA1 gene ampliconwas tagged with Quasar 670 fluorophore and BHQ-2 quencher, BmTPKamplicon with CAL Fluor Orange 560 fluorophore and BHQ-1 quencher andAPH1387 amplicon using CAL Fluor Red 610 and BHQ-2 quencher. The lengthsof the probe sequences were chosen so that they would form a stablehybrid with the target at 5 to 10° C. above the annealing temperature(60° C.) of the PCR assay. The 5′ and 3′ arm sequences of the molecularbeacons were designed to form a stable hybrid at 5 to 10° C. above theannealing temperature of the PCR assay. The fluorophores and quencherswere chosen based on the specifications of the spectrofluorometricthermal cycler platform on which the assays were carried out and theircompatibility in one multiplex assay. The sequences of the molecularbeacons used in this study are listed in Table 1. A detailed protocolfor the synthesis and purification of molecular beacons can be found atmolecular-beacons.org. For this study, molecular beacons were orderedfrom Biosearch Technologies, CA. Initial standardization of PCRconditions was conducted by using SYBR Green I dye (Life Technologies,CA) and was followed by replacing SYBR Green with specific molecularbeacon probes in the assays.

Real-time PCR. Since genome sizes of B. burgdorferi and human are 1.5 Mband 3.2 Gb respectively, 2 ng of B. burgdorferi genomic DNA containsapproximately 10⁶ copies of recA gene, while 350 ng of human genomic DNAcontains approximately 10⁵ copies of ACTA1 gene. Similarly, genome sizesof B. microti and A. phagocytophilum are 6.5 Mb and 1.47 Mb,respectively. Therefore, 10⁶ copies of thiamine pyrophosphokinase geneof B. microti (BmTPK) and APH1387 are calculated to be present in 8 ngand 2 ng, respectively. All primer and probe sequences are listed inTable 1. A 222 bp fragment from recA gene of B. burgdorferi using RecFand RecR primers and a 104 bp fragment from human alpha actin A1 (ACTA1)gene using 5ACTA1 and 3ACTA1 primers were amplified by PCR in 0.2 mloptical tubes using a Bio-Rad CFX96 Touch Real-time PCR system (Bio-RadLife-Science Research, CA). Amplification was performed in 25 μlreaction mixtures containing Amplitaq Gold PCR reaction buffer (LifeTechnologies) supplemented with 3 mM MgCl2, 500 ng/μl of bovine serumalbumin, 250 μM of each deoxynucleoside triphosphate (dNTP), 0.5 μM ofeach set of primers and 5U of Amplitaq Gold polymerase. For eachamplification reaction, 5 μM of the sample was used to minimize thevariation due to pipetting error. BmTPK gene of B. microti and APH1387gene of A. phagocytophilum were amplified using the primers and clonedin TopoXL vector from Invitrogen to optimize conditions such that thesetwo pathogens can be detected under the same conditions as Lymespirochetes. Amplification of a 141 bp amplicon from BmTPK gene using5BmTPK and 3BmTPK primers and a 152 bp amplicon of APH1387 gene using5Aphagocyt and 3Aphagocyt primers were carried out. Molecular beaconprobes, BmTPK and APH1387 were used for detection of the BmTPK andAPH1387 amplicons, respectively. Data were processed using the softwareprovided by the manufacturer.

For quadruplex real-time PCR assays, genomic DNA of B. burgdorferi andhuman, and clones of BmTPK and APH1387 were used as templates, and 500nM each of RecF and RecR primers and 5BmTPK and 3BmTPK primers, 250 nMeach of 5Aphagocyt and 3Aphagocyt primers, 100 nM each of 5ACTA1 and3ACTA1 primers, 25 ng each of RecA3, BmTPK, APH1387, and ACTA1 molecularbeacons were included in each reaction. The amplification programconsisted of initial heating at 95° C. for 5 minutes, followed by 60cycles of heating at 95° C. for 15 s, annealing and fluorescencedetection at 60° C. for 30 s, and polymerization at 72° C. for 20 s. Allassays were performed with a Bio-Rad CFX96 Touch Real-time PCR DetectionSystem.

For confirmation of the quadruplex assay in which plasmids containingBmTPK and APH1387 were used, we incorporated different concentrations ofgenomic DNA of B. burgdorferi, B. microti (6.5 Mb) and A.phagocytophilum (1.47 Mb) in the triplex real time-PCR. Human DNAcontrol was not included in these assays. By using different relativegenomic copy numbers using the conditions similar to those describedabove for quadruplex assay, these confirmatory assays (FIGS. 6 and 7)validated our assay for simultaneous detection of all three pathogens.

To differentiate three major species that cause Lyme disease in Europe,B. burgdorferi, B. afzelii and B. garinii, asymmetric PCR assay wasperformed in 25 μl volume such that primer synthesizing the targetstrand of molecular beacon was used in excess. The primers for recA genethat are from the conserved region in all three species, RecF3 and RecR3were designed to amplify a slightly longer, 287 bp fragment in thisasymmetric PCR assay. The reaction mixture contained 1× Amplitaq GoldPCR buffer supplemented with 3 mM of MgCl2, 500 ng/μl of bovine serumalbumin, 250 μM of each dNTP, 30 nM of RecF3 primer, 1000 nM of RecR3primer, 12.5 ng of RecA3 molecular beacon and 5 units of Amplitaq Goldpolymerase. The amplification program consisted of initial heating at95° C. for 5 minutes, followed by 60 cycles of heating at 95° C. for 15s, annealing and fluorescence detection at 60° C. for 30 s, andpolymerization at 72° C. for 20 s. It was immediately followed byincubation at 25° C. for 2 minutes to allow annealing, and then a meltcurve was included by increasing temperature from 25° C. to 95° C. in 1°C. step, with each step lasting 2 minutes while monitoring thefluorescence. For analysis, the first derivative of the denaturationprofile was determined as described previously [El-Hajj et al. (2009) JClin Microbiol 47:1190-1198].

TABLE 1 Sequence of PCR primers and molecular beacon probes PCR Size ofprimers/ Tm PCR SEQ Fluorophore/ Probes Sequence Length (° C.) ampliconID No. Quencher RecF 5′ GTG GAT CTA TTG 30 66.1 222 bp 10TAT TAG ATG AGG CTC TCG 3′ RecR 5′ GCC AAA GTT CTG 30 67.3 11CAA CAT TAA CAC CTA AAG 3′ RecA3 5′ CTG GCG GAT ATC 26 5 FAM/CTA GGG GG CGC BHQ-1 CAG 3′ RecF3 5′ GCA AGA GTT 20 53.7 12CAA ATA GAA AA 3′ RecR3 5′ AAA GCT TTT GCA 19 54.7 13 TAA ACA G 3′5BmTPK 5′ TGA GAG GAA 19 61.4 141 bp 14 CGA CCA TAG C 3′ 3BmTPK5′ CCA TCA GGT AAA 22 61.6 15 TCA CAC GAA A 3′ BmTPK 5′ CGC GTC GGT GTT35 16 CAL Fluor GTT GAC CAG CGG Orange 560/ CCG CG GAC GCG 3′ BHQ-15Aphagocyt 5′ ATG GCT ACT ACG 18 57.9 152 bp 18 AAG GAT 3′ 3Aphagocyt5′ CGA AGC AAC 19 58.0 19 ATC TCT ACA T 3′ Aph1387 5′ CGG TGC GAC 36 20CAL Fluor AAA GAT GCC AGC Red 610/ ACT AAT GCG GCA BHQ-2 CCG 3′ 5ACTA15′ AGA GCA AGA 18 58.0 104 bp 21 GAG GTA TCC 3′ 3ACTA15′ CTC GTT GTA GAA 18 57.7 22 GGT GTG 3′ ACTA1 5′ CGC TGC CCT ATC 35 23Quasar 670/ GAG CAC GGC ATC BHQ-2 ATC AC GCA GCG 3′ Bb-RecA35′TTATGCGCCCCCT 30 2 target AGGATATCCGCCAA TGC3′ 5BmicrotiT5′ AAT ATT GTT GAA 26 64.2  600 bp 24 PK TGG GGA TAT TTG TG 3′3BmicrotiT 5′ AAT AAT ATA GCT 31 60.2 25 PK TTT CCA AAA TAT AAC TGA C 3′5ApAPH13 5′ ATG TAT GGT ATA 29 57.8 1737 bp 26 87 GAT ATA GAG CTAAGT GA 3′ 3ApAPH13 5′ CTA ATA ACT TAG 29 62.2 27 87 AAC ATC TTC ATCGTC AG 3′ Ba-RecA3 5′TTATGCGCCCCCT 30 3 target AGGATATCCACCAA TGC 3′Bg-RecA3 5′TTATTCGCCCCCTA 30 4 target GGATATCCACCAAT GC 3′

In the sequences of the molecular beacon probes, the nucleotides of thecomplementary arms are underlined, and the nucleotides of thesingle-strand loops are bolded.

Borrelia burgdorferi genomic DNA

-   -   1.52×10⁶ bp (chromosome+plasmids)    -   6.0×10⁵ copies/ng    -   2 ng≠10⁶ copies

Babesia microti genomic DNA

-   -   6.5×10⁶ bp (chromosomes)    -   1.4×10⁵ copies/ng    -   8 ng≈10⁶ copies

Anaplasma phygocytophilum genomic DNA

-   -   1.47×10⁶ bp (circular genome)    -   6.2×10⁵ copies/ng    -   2 ng≈10⁶ copies

Human HEK293 cells genomic DNA

-   -   3.2×10⁹ bp (chromosomes)    -   285 copies/ng    -   350 ng≈10⁵ copies

Example 1

This example demonstrates that in an assay according to this invention,molecular beacons can detect B. burgdorferi between 1 and 10⁶ startingcopies and can quantify the starting copy number in a multiplex assay,when a human DNA sequence is also amplified and detected in real time.Real-time amplification plots of recA and ACT A1 gene target sequencesin PCR assays to estimate quantities of B. burgdorferi (FIG. 1A) andhuman (FIG. 1C) DNA are shown. Human DNA (containing 10⁵ ACT A1 copies)spiked with ten-fold dilutions of B. burgdorferi strain N40 ranging from1 to 10⁶ were used in the PCR assays containing both RecA3 and ACTA1molecular beacons. Sensitivity and specificity of the detection systemis indicated by the ability of RecA3 and ACTA1 molecular beacons toquantify the amplicons from both the recA and the ACT A1 genes in thesame PCR assay tubes. A high coefficient of correlation (r2=0.999)between the PCR threshold (Ct) values and the spirochete number obtainedfrom the standard curve (FIG. 1B) demonstrates that a multiplex assayaccording to this invention can be used effectively to quantifyspirochete burden in infected tissues using multiplex assay system. Thehuman DNA target sequence is not critical. We have chosen a sequence ofthe ACT A1 gene, but other unique human DNA targets could also be usedby designing a suitable primers pair and respective molecular beaconprobe.

Real-time PCR detection of recA amplicon of B. burgdorferi in thepresence of human genomic DNA. Molecular beacons and PCR conditions wereoptimized for quantitative detection of B. burgdorferi DNA by real-timePCR (102). To use the assay for diagnosis of Lyme disease in thepatients, it is important that it works in the presence of human genomicDNA. Therefore, the same quantity of human DNA (350 ng genomic DNA or10⁵ ACT A1 copy number) was spiked with a ten-fold dilution series ofgenomic DNA of B. burgdorferi, from 10⁶ copies to 10° copies. Sincesimultaneous detection of pathogen and host PCR products is possibleusing molecular beacons tagged with different fluorophores,normalization of the host DNA in patient sample is more convenient andaccurate. In addition, accurate detection of host DNA in each sampleensures the quality of the DNA preparation. To evaluate this premise,primers and molecular beacons for both recA amplicon of B. burgdorferiand ACT A1 amplicon of human DNA were included in the startingamplification reaction mixtures, along with the B. burgdorferi genomicDNA and human genomic DNA.

Amplification plots of the recA gene in the PCR assays (FIG. 1A), asdetected by fluorescence intensity at the end of each cycle at theannealing temperature, show that the presence of 1 to 10⁶ spirochetes isdetected using the RecA3 molecular beacon. A standard curve (FIG. 1B)generated by plotting the log of the known initial copy numbers of B.burgdorferi versus the threshold (Ct) values from FIG. 1A indicates thatthe threshold cycle is inversely proportional to the number of targetmolecules present in the samples. A high coefficient of correlation(r2=0.999) between the B. burgdorferi copy number and the thresholdcycle number (Ct) obtained from the standard curve indicates that thiscurve can be used to determine the quantity of spirochetes in infectedmouse tissues. Since identical Ct values for ACT A1 in all samples weredetected as expected, the number of copies of B. burgdorferi genome inthe sample or the presence of human DNA does not affect sensitivity ofdetection of amplicon of pathogens and the host in multiplex assay(FIGS. 1A and 1C).

Example 2

This example demonstrates that denaturation profiles of the hybrids ofRecA3 molecular beacon probe with the target oligos from three Borreliaspecies can distinguish B. burgdorferi, B. afzelii and B. garinii (FIG.2A). Additionally, one real-time PCR assay distinguishes three majorLyme spirochetes using post-amplification denaturation curves. Either amelting curve made by slowly increasing the reaction temperature or anannealing curve made by slowly decreasing the reaction temperature isused. Using different sets of primers and the same RecA3 molecularbeacon probe, asymmetric real-time PCR amplified the fragment of recAgene from all three Borrelia species, and a post-PCR denaturationprofile could distinguish all three spirochete species (B. burgdorferi,B. afzelii, and B. garinii) from one another based on the Tm of theprobe-target hybrid (FIG. 2B). In the normal real-time PCR, either (i)competition between the probe and complementary strand, or (ii)secondary or tertiary structure of the target strand may decreaseformation of the probe-target strand. To overcome this, non-symmetricPCR methods in which one primer or a primer pair is present in limitingamount, including LATE-PCR (Quan, P. L. et al., 2008, Antiviral Res:79:1-5) or asymmetric PCR, was conducted such that primers for thecomplementary and target strand were used at 30 nM versus 1000 nMconcentrations and lower amount of the molecular beacon RecA3 (12.5 ng)was included in the reaction mixture. It is noted that during PCRamplification in which real-time fluorescence intensity is monitoredduring the PCR annealing step (here 50° C.) as a function of cyclenumber (see FIG. 1A), B. burgdorferi gave the strongest signal, B.afzelii gave a somewhat weaker signal, and B. garinii gave the weakestsignal, as shown by the curves in FIG. 2. Example 2 and FIG. 2A alsoindicate that the molecular beacon probe could be made perfectlycomplementary to either the B. afzelii or B. garinii sequence, in whichcase that species would have the highest Tm and give the strongestreal-time signal, and B. burgdorferi have a lower Tm and give a weakersignal.

Differentiation of Lyme spirochete species using the denaturationprofiles. Only a few single nucleotide polymorphisms (SNPs) are presentin the amplicon sequences of B. burgdorferi sensu stricto with 100%match and corresponding B. afzelii and B. microti sequences. The loopsequence of the RecA3 molecular beacon (SEQ ID No. 5), 3′ GACCGCGGGGGATCCTATAGGCG GTC 5′, is perfectly complementary to the B.burgdorferi 5′ TTAT GCGCCCCCTAGGATATCCGCCA ATGC 3′ (SEQ ID No. 2) butless than perfectly 5′ TTAT GCGCCCCCTAGGATATCCACCA ATGC 3′ (SEQ ID No.3) and less than perfectly complementary to the species B. garinii 5′TTAT TCGCCCCCTAGGATATCCACCA ATGC 3′ (SEQ ID No. 4).

In order to determine the melting temperatures of the molecular beaconstem and the molecular beacon probe-target hybrid, a denaturationprofile analysis was carried out. Three tubes containing 200 nMmolecular beacon RecA3, 3 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH8.0), in a 50-μl volume were prepared. A two-fold molar excess of anoligonucleotide that is complementary to the molecular beacon probesequence, either B. burgdorferi, B. afzelii, or B. garinii sequencestated above, or only buffer were added in these tubes. The fluorescenceof each solution was determined as a function of temperature. Thethermal cycler was programmed to generate a denaturation curve, that is,to increase the temperature of the solutions from 40° C. to 90° C. in 1°C. steps, with each step lasting 1 min, while monitoring fluorescenceduring each step. The RecA3 molecular beacon was able to distinguish allthree species by determination of the melting temperature (Tm) of thehybrids formed by the binding of these molecular beacons to the targetsequences (FIG. 2A).

Using the asymmetric PCR, each target sequence was amplified usingdifferent primers, in this case RecF3 and RecR3 primers, and RecA3molecular beacon (Table 1), followed by melt analysis. Taking advantageof the SNPs as discussed in the preceding paragraph, melting curveanalysis identified DNA of three species. As shown in FIG. 2B, themelting-curve profiles and the Tm's (66° C., 59° C. and 55° C.) clearlyidentified which species was present. Viewed as denaturation profiles,one sees that as the temperature of the solution containing the hybridsis slowly raised, dissociation of the molecular beacon probes from thetarget strands is observed as a decrease in fluorescence intensity dueto the conformational reorganization of the molecular beacons intoquenched hairpin structures. The results of these experiments indicatethat the three species can be distinguished from each other by simplydetermining the stability (as expressed by the Tm) of the resultinghybrids. These results indicate that one can use hybrid melting curvesto identify more than one species of Lyme spirochete in clinical sampleswhen each is present in 10% or more of the total spirochete populationin a sample.

Example 3

This example demonstrates that in assays of this invention, molecularbeacons can detect DNA from 1 and 10⁶ Babesia microti in a multiplexassay in the presence of human DNA and can quantify the starting copynumber in a multiplex assay, when a human DNA sequence is also amplifiedand detected in real time. Amplification plots of BmTPK and ACT A1 genesin PCR assays using the human DNA representing 10⁵ ACT A1 copies spikedwith ten-fold dilutions from 1 to 10⁶ of B. microti DNA copies were usedto estimate quantities of B. microti (FIG. 3A) and human (FIG. 3C) DNAby employing both BmTPK and ACTA1 molecular beacons. The assayquantified amplicons from both the BmTPK and the ACT A1 genes in thesame PCR assay tubes. A high coefficient of correlation (r2=0.993)between the Ct values and the parasite numbers obtained from thestandard curve (FIG. 3B) indicates that the molecular beacons can beused effectively to quantify the parasite burden in the human infectedcells using multiplex assay system using the optimized conditions.

TPK gene amplicon of B. microti can be detected efficiently along withhuman ACT A1 amplicon in the multiplex assay. Two enzymes wereidentified to be important in central metabolism of B. microti by genomesequencing of this parasite [Cornillot et al. (2012) Nucleic Acids Res40:102-114], Lactate dehydrogenase (LDH) and TPK. Only LDH is expressedduring intra-erythrocytic multiplication stage of this pathogen. BothLDH and TPK genes were cloned and initially both of the plasmid cloneswere used as templates for real-time PCR using SYBR green and respectivemolecular beacons. However, only BmTPK showed promising results.Therefore, extensive investigation was conducted using the cloned BmTPKgene only [primers 5BmicrotiTPK (SEQ ID No. 24) and 3BmicrotiTPK (SEQ IDNo. 25)]. Ten-fold dilutions of plasmid containing BmTPK gene, startingwith 10⁶ copies, were prepared in the human DNA (350 ng) containing 10⁵copies of ACT A1 to use as template. Using 5BmTPK and 3BmTPK primers,BmTPK molecular beacon and PCR conditions described in the methodssection, TPK and ACT A1 amplicons were detected in real time andquantified. Copy number from 10⁶ to 10° of BmTPK showed steady increasein threshold cycle number (FIG. 3A). In other experiments single copynumber sometimes was indistinguishable from the curve when 10 copies ofTPK were present. These results are also depicted in the standard curve(FIG. 3B) and are reflected in the coefficient of correlation(r2=0.993). Overlapping ACT A1 detection curves indicate that the sameconcentration (10⁵ copies) of human DNA included in different tubes forTPK-containing plasmids dilutions had the same threshold cycle and sowere accurately quantitated. Thus, it is expected that 10 copies of TPKwill be detected consistently in this assay.

Example 4

This example demonstrates that in assays of this invention, molecularbeacons can detect DNA from 1 and 10⁶ Anaplasma phagocytophilum in amultiplex assay in the presence of human DNA and can quantify thestarting copy number in a multiplex assay, when a human DNA sequence isalso amplified and detected in real time. Amplification plots of APH1387and ACT A1 genes in PCR assays using the human DNA representing 10⁵ ACTA1 copies spiked with ten-fold dilutions from 1 to 10⁶ plasmid copiescontaining APH1387 were used to estimate quantities of A.phagocytophilum (FIG. 4A) and human (FIG. 4C) DNA by employing bothAph1387 and ACT A1 molecular beacons. The assay quantified ampliconsfrom both the APH1387 and the ACT A1 genes in the same PCR assay tubes.A high coefficient of correlation (r2=0.985) between the Ct values andthe parasite numbers obtained from the standard curve (FIG. 4B)indicates that the molecular beacons can quantify burden of thisintracellular pathogen in the human infected cells using multiplex assaysystem using the standardized conditions in a sensitive and specificmanner.

Specific detection of APH1387 amplicon in the presence of human DNAusing molecular beacon probes in multiplex assay. A. phagocytophilum isan obligate intracellular pathogen that multiplies within a vacuoleinside the host cells that avoids fusion with lysosome. APH1387 of A.phagocytophilum was identified as the first protein that localizes tothe vacuolar membrane containing this pathogen [Huang et al. (2010) JMicrobiol 48:877-880]. Since the gene is uniquely present in A.phagocytophilum and is highly conserved in various strains, we selectedit to use for detection of this bacterium by rt-PCR and first amplifiedit using 5ApAPH1387 and 3ApAPH1387 primers (SEQ ID Nos. 26 and 27). Byusing the strategy used for TPK gene containing plasmid for B. microtias described above, APH1387 containing plasmid was diluted in human DNA,and PCR was conducted using 5Aphagocyt and 3Aphagocyt primers andAph1387 molecular beacon. Primers for human ACT A1 amplicon and ACTA1molecular beacon were also included in the reaction mixture. Conditionsfor PCR were identical to those used for Lyme spirochetes recA and B.microti TPK gene amplifications. Real-time fluorescence readings of theAph1387 probe are presented in FIG. 4A. Real-time readings of the ActA1probe are presented in FIG. 4C. Because the curves for 10¹ copies oftarget and 10° copies of target were not well separated, the APH1387detection limit was judged to be close to 10 (FIG. 4A), similar to thatfor the TPK detection sensitivity of B. microti observed sometimes andnot close to 1 consistently as observed for recA amplicon of Lymespirochetes. Again, the results were reflected in the standard curve andcoefficient of correlation of 0.985 (FIG. 4B). Sensitivity of detectionof human ACT A1 amplicon was maintained similar to the multiplex assaysdescribed for recA and BmTPK amplicons above.

Example 5

This example describes a quadruplex assay according to this invention.Example 5 demonstrates that inclusion of three tick-borne pathogens inthe presence of human DNA in a single quadruplex assay does not affectthe sensitivity of their detection. Conditions for a quadruplex PCRassay were optimized such that eight primers (four pairs) and fourdifferent molecular beacons for respective amplicons were present in thesame tube along with the other reagents required for PCR. Sensitivity ofdetection of two bacterial pathogens, extracellular spirochete B.burgdorferi (FIG. 5A) and obligate intracellular pathogen A.phagocytophilum (FIG. 5C), along with the intracellular parasite, B.microti (FIG. 5B), was not affected in this quadruplex assay,demonstrating that the assay can be extended for simultaneous diagnosisof all three tick-borne pathogens in patients, especially in endemicregions. Detection of the ACT A1 amplicon in the same reaction willoffer as control for human DNA (FIG. 5D) and quality of DNA preparationwhen the patient samples will be used for diagnosis of the infectingorganism. Since most real time PCR machines can now detect fivefluorophore simultaneously, this assay can be expanded to includeanother tick-borne pathogen, such as, Powassan virus, which is emergingin Hudson Valley ticks and cause rather fatal disease.

Optimization of conditions for simultaneous detection of recA of Lymespirochetes, TPK of B. microti and APH1387 amplicon of A.phagocytophilum. Since Lyme disease is the most prevalent tick-bornedisease in the USA and it is also widespread in Europe, and becauseemerging pathogens Babesia species and A. phagocytophilum are found tobe co-infecting the same species of the ticks, it is very likely that afew cases of co-infections in humans will increase steadily in the nearfuture. Therefore, development of a single multiplex real-time PCR assayfor detection of all three tick-borne pathogens simultaneously in thepatient samples in a sensitive and specific manner is expedientlywarranted. To achieve this goal, experimental conditions werestandardized such that genomic DNA of B. burgdorferi and plasmidscontaining BmTPK and APH1387 genes were serially diluted in human DNAcontaining 10⁵ copies of ACT A1 gene. The quadruplex reaction mixturewas amplified by PCR, with probe readings in real time. FIG. 5A showsthe results for varying concentrations of the recA target, FIG. 5B showsthe results with varying concentrations of the BmTPK target, FIG. 5Cshows the results with varying concentrations of the Aph1387 target, andFIG. 5D shows the results for the ACT A1 target (10⁵ copies). Byincreasing the concentration of molecular beacons in the quadruplexassay mixture, we were able to improve the sensitivity of detection ofboth B. microti TPK and A. phagocytophilum APH1387 amplicons such thatcurve for 1 copy was clearly distinguishable from 10 copies (FIGS. 5Band 5C). Amplicons from all three pathogens along with the control humanACT A1 gene amplicon were detected in one assay. Sensitivity ofdetection of any of the three pathogens amplicons was not affected inthe assay.

Example 6

This example demonstrates that in a multiples assay an excess of B.burgdorferi genomic DNA does not affect sensitivity of detection of A.phagocytophilum and B. microti genomic DNA. Genomic DNA of B.burgdorferi representing 10⁶ copies of recA gene mixed with A.phagocytophilum and B. microti genomic DNA each reflecting 10³ (FIG. 6)copies of BmTPK and APH1387 genes, respectively were used as template inthe multiplex PCR amplification assay. Sensitivity and specificity ofdetection of BmTPK and APH1387 amplicons was retained in a 10³-foldexcess of recA gene copies in the reaction mixture.

Sensitivity of detection of emerging pathogens B. microti and A.phagocytophilum DNA is retained in the presence of excess of B.burgdorferi DNA. Even though cloned genes of both of these pathogens inplasmids could be detected and quantitated when present individually andtogether with B. burgdorferi, it is essential to determine if thesensitivity is maintained when their genomic DNA is used as template. Inaddition, quantities of these emerging pathogens may vary in the patientsamples. Therefore, the sensitivity of the assay for detection of B.microti and A. phagocytophilum in excess of B. burgdorferi DNA wasassessed. B. burgdorferi genomic DNA/recA copy number (10⁶) along withgenomic DNA equivalent to 10³ copies of BmTPK and APH1387 each was used.Real-time probe readings are presented in FIG. 6. As shown by thisFigure, the accuracy and sensitivity of detection of B. microti and A.phagocytophilum was not affected by 10³-fold excess of B. burgdorferigenomic DNA, validating the applicability of multiplex assays accordingto this invention for diagnosis of all three tick-borne infections evenif Lyme spirochetes are present in excess in the sample, such as insynovial fluid or skin biopsy samples.

Example 7

This example demonstrates that in a multiplex assay with an excessiveDNA quantity, in this case a one hundred-fold excess, of A.phagocytophilum and B. microti genomic DNA relative to the B.burgdorferi DNA does not affect sensitivity of detection of spirochetalrecA amplicon. Genomic DNA of B. burgdorferi representing 10 copies ofrecA gene mixed with A. phagocytophilum and B. microti genomic DNA eachreflecting 10³ copies of BmTPK and APH1387 genes, respectively were usedas template in the multiplex assay. Real-time fluorescence curves forthe three probes are shown in FIG. 7. As FIG. 7 demonstrates,sensitivity and specificity of detection of recA amplicon was notaffected by the excess of A. phagocytophilum and B. microti genomic DNAin the reaction mixture. Such a scenario is expected in nature,particularly in the blood samples of patients co-infected with thesetick-borne pathogens.

B. burgdorferi can be accurately detected even in the 100-fold excess ofB. microti and A. phagocytophilum genomic DNA. Blood is primarily usedas a conduit by Lyme spirochetes to disseminate to various tissues suchthat usually only a few B. burgdorferi are present in the blood at anygiven time. Therefore, it is likely that blood-borne pathogens A.phagocytophilum and B. microti could be present in higher numbers inblood during co-infection with B. burgdorferi. To determine whetherdetection of B. burgdorferi could be affected by the presence of higherlevels of parasitemia by B. microti and/or bacteremia by A.phagocytophilum, genomic DNA of all three pathogens were mixed such thatthe copy number of BmTPK and APH1387 (10³) was 100-fold of that of recAof B. burgdorferi [Bakken (2002) J Contin Educ Health Prof 22:131-141]in a triplex PCR amplification reaction containing primers and a probefor each of the three genes. Real-time curves (fluorescence intensityversus PCR cycle number) from each of the three probes are shown in FIG.7. Ten copies of B. burgdorferi recA per one thousand copies of BmTPKand APH1387 were consistently detected in a multiplex assay (FIG. 7).These results demonstrate that irrespective of the levels of eachpathogen relative to the other two pathogens, multiplex rt-PCR assaysaccording to this invention accurately detect each pathogen in themixture.

Example 8

This example describes a hexaplex assay according to this invention.This example demonstrates that the assay can simultaneously in real timedetect five tick-borne pathogens: three species of Lyme diseasespirochetes (B. burgdorferi, B. afzelii, and B. garinii), the protozoanpathogen B. microti, and the intracellular bacterial pathogen A.phagocytophilum, as well as detecting the presence of human ACT A1 DNA,in a single assay.

Conditions for the hexaplex PCR assay are optimized in the mannerdescribed above, such that four pairs of primers and six differentlycolored molecular beacons are included in the same tube, along with theother reagents required for PCR. One pair of primers (listed in Table 1)enables the amplification of a region of Borrelia DNA that is common toall three Borrelia species, and three different molecular beacons arepresent, each specific for a different Borrelia species, and eachlabeled with a differently colored fluorophore. The three other pairs ofprimers (also listed in Table 1) enable the amplification of a region ofA. phagocytophilum DNA, a region of Borrelia microti (or other Babesiaspecies) DNA, and a region of the human ACT A1 gene, and threeadditional molecular beacons are present, each specific for one of thethree resulting amplicons, and each labeled with a differently coloredfluorophore. The real-time PCR reaction and detection of thefluorescence from each of the six molecular beacons is carried out inthe manner described above in an instrument that is able to distinguishat least six different colors. All five pathogens, along with thecontrol human ACT A1 gene, are therefore detected and quantitated in asingle real-time PCR assay,

The optimized multiplex assays of the present invention accuratelydetect and quantify a single spirochete recA gene copy spiked in thehuman DNA. The presence of high concentrations of human genomic DNA(containing 10⁵ copies of ACT A1 gene) did not affect accuracy of theassay (FIG. 1) as also shown by almost perfect coefficient ofcorrelation (r2=0.999) between threshold cycle and copy number of B.burgdorferi DNA. Consistency of detection of ACT A1 gene amplicon in alltubes indicated that each molecular beacon probe, for B. burgdorferi andACTA1, tagged with a different fluorophore can accurately detectrespective amplicons, and the sensitivity of the assay is not affecteddue to interference from the presence of other pathogens DNA. As proofof the principle, specific oligonucleotides for B. burgdorferi sensustricto, B. afzelii and B. garinii sequence were designed which show thepresence of SNPs in the probe-binding region. Using a single molecularbeacon, three species oligos were discriminated by determining the Tmfrom the denaturation curves (FIG. 2). Real-time PCR using the hbb geneand reverse complementary 3′-fluorescein-labeled probe designed againstthe forward strand of amplicon was able to distinguish differentBorrelia species by Tm determination [Ferdin et al. (2010) J MicrobiolMeth 82:115-119]. The methods of the present invention accurately detectLyme spirochetes and also distinguish three different species of Lymespirochetes. This assay can be easily extended to include other emergingBorrelia species implicated in Lyme disease, such as B. miyamotoi, whichappears to cause more severe illness in humans [Krause et al. (2013) NewEngland J Med 368:291-293]. Such discrimination is important todetermine accurate association between specific chronic Lyme diseasesymptoms with particular Borrelia species.

The best time to develop an efficient diagnostic test is when infectionsby a particular organism start emerging among human or animalpopulations, environment or in the vectors. Since infections of ticks bytwo tick-borne pathogens, A. phagocytophilum and Babesia species, havebeen increasing in both Europe and the USA, and because the cases bythese emerging pathogens are also being reported in higher numbers onboth continents [Beugnet and Marie (2009) Veterinary Parasitology163:298-305; Dantas-Torres et al. (2012) Trends in Parasitology28:437-446; Graham et al. (2011) Pediatr Emerg Care 27:141-147, quiz148-50; Heyman et al. (2010) Expert Rev Anti Infect Ther 8:33-50; andSocolovschi et al. (2009) Parasitology 16:259-273], the presentinvention was expanded to include detection of these two pathogens.Indeed, co-infections with these tick-borne pathogens have startedappearing in patients, resulting in exhibition of more severe illnesses[Horowitz et al. (2013) Clinical infectious diseases 56:93-99; andWormser et al. (2013) J Clin Microbiol 51:954-958]. Optimized conditionsfor detecting each emerging pathogen, using the B. microti and A.phagocytophilum genes BmTPK and APH1387, respectively, with human ACT A1individually (FIGS. 3 and 4), worked well, even in a quadrupex assay inwhich serially diluted genomic DNA of B. burgdorferi and human could beaccurately detected in addition to BmTPK and APH1387 containing plasmidDNA (FIG. 5). Moreover, this test detected as few as 10³ copies of bothAPH1387 and BmTPK in mixed genomic DNA in the presence of an excess(10³-fold copy number) of B. burgdorferi DNA, confirming the sensitivityand accuracy of the assay. The methods of the present inventiondemonstrate an efficient and quick assay to detect individual pathogens,such as B. microti in blood bank samples using the approach shown inFIG. 3. Co-infections with two or three pathogens in the endemic regionsfor these tick-borne diseases using the triplex or quadruplex assay arealso diagnosed according to the methods of the present invention (FIGS.5, 6, and 7). The present invention describes novel assays for thesensitive detection of three tick-borne pathogens simultaneously. Theseassays can be easily adapted for the patient samples in the future withlittle modification if needed.

The foregoing examples and description of the preferred embodimentsshould be interpreted as illustrating, rather than as limiting thepresent invention as defined in the specification. As will be readilyappreciated, numerous variations and combinations of the features setforth above can be utilized without departing from the present inventionas set forth in the claims. Such variations are not regarded as adeparture from the scope of the invention, and all such variations areintended to be included within the scope of the following claims. Allreferences cited herein are incorporated by reference in theirentireties.

1. A multiplex primer-dependent nucleic acid amplification and detectionmethod for diagnosing at least two tick-borne diseases from the groupconsisting of Lyme, babesiosis and anaplasmosis in a sample from a humanor other mammal, comprising the steps of: a. contacting DNA from saidsample with primer-dependent amplification reagents that include atleast two of the following: (i) a primer pair for a recA gene sequenceof Borrelia that differs among B. burgdorferi, B. afzelii, and B.garinii, and one or a complement of Borrelia-specific molecular beaconprobes capable of distinguishing among those species; (ii) a primer pairfor a conserved BmTPK gene sequence of B. microti (a conserved andspecific gene present among different Babesia species and strains andthat encodes a protein critical for central metabolism of the parasite),and a Babesia-specific molecular beacon probe; and (iii) a primer pairfor a conserved APH 1387 gene sequence of A. phagocytophilum (which isunique and encodes for a protein critical for pathogenesis of thisobligate intracellular pathogen and hence, present among differentstrains of this bacterium) sequence of A. phagocytophilum, and anAnaplasmosis-specific molecular beacon probe, wherein said primer pairsdefine amplicons that are 70-300 base pairs in length, and wherein eachdifferent molecular beacon probe is labeled with a spectrallydistinguishable fluorescent or luminescent signaling moiety, b.performing said primer-dependent amplification to obtain amplifiedproducts of said gene sequences, and c. detecting said amplifiedproducts, thereby identifying said subject as infected or not infectedwith one or more of said at least two pathogens.
 2. The method of claim1, wherein said detection includes real-time detection of said amplifiedproducts.
 3. The method of claim 1 wherein the primer-dependentamplification method is a polymerase chain reaction (PCR) method.
 4. Themethod of claim 1 wherein the amplification reagents include both (ii)and (iii).
 5. The method of claim 4 wherein the molecular beacon probefor the BmTPK gene has the single-stranded loop region (SEQ ID No. 28)of SEQ ID No. 6, and the molecular beacon probe for the APH 1387 genehas the single-stranded loop region (SEQ ID No. 29) of SEQ ID No.
 9. 6.The method of claim 1 wherein the PCR reagents include a primer pair anda molecular beacon probe for a human DNA gene sequence, and saiddetection includes detecting amplicon from amplification of said humanDNA gene sequence.
 7. The method of claim 6 wherein the human DNA genesequence is a 70-300 base-pair region of the ACT A1 gene.
 8. The methodof claim 1 wherein the PCR reagents include a primer pair (SEQ ID Nos.12 and 13) and a single molecular beacon Borrelia probe for said recAgene sequence of Borrelia that hybridizes to amplified product from allthree species B. burgdorferi, B. afzelii, and B. garinii but withdetectably different melting temperatures, and said detection includesdetermining the melting temperature of probe-target hybrids for theBorrelia probe following amplification.
 9. The method of claim 8 whereinthe molecular beacon probe for the recA gene has the single-strandedloop region (SEQ ID No. 30) of SEQ ID No. 5.